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. 2012 Jul 24;287(37):31172–31184. doi: 10.1074/jbc.M112.387704

FIGURE 6.

FIGURE 6.

MHC I saturation of the PLC varies with allele. A, C1R.A2-His cells (black line) were labeled with BB7.2 (anti-HLA-A2) and FITC-conjugated goat anti-mouse IgG and analyzed for MHC I surface expression in comparison with C1R cells (gray line) by flow cytometry (left panel). C1R and C1R.A2-His digitonin lysates were also assessed for His and endogenous HLA-C4 expression by SDS-PAGE and immunoblotting with mouse anti-His (top right panel) and HC10 (anti-HLA-C HC) (bottom right panel), respectively. Anti-GRp94 was used as a loading control. C1R (B) and C1R.A2-His (C) digitonin lysates were sequentially immunoprecipitated with PaSta2, followed by a fifth immunoprecipitation with PaSta1 or PaSta2. After SDS-PAGE and immunoblotting with R.Gp48C (anti-tapasin), R.RING4C (anti-TAP1), and 3B10.7 (anti-MHC I HC) (for C1R) or rabbit anti-His and HC10 (anti-HLA-C HC) (for C1R.A2-His), the bands were quantitated. Both HLA-A2-His and HLA-C4 (left panel) and total MHC I (right panel) were plotted for C1R.A2-His. These results are expressed as the means plus the S.D. of two independent experiments. White bars, tapasin-ERp57. Gray bars, TAP1. Black bars, total MHC I. Horizontally striped bars, HLA-A2-His. Vertically striped bars, endogenous HLA-C4.